AIMS:Cardiovascular disease is the leading cause of death for individuals diagnosed with type II diabetes mellitus (DM). Changes in cardiac function, left ventricular wall thickness and fibrosis have all been described in patients and animal models of diabetes; however, the factors mediating increased matrix deposition remain unclear. The goal of this study was to evaluate whether cardiac fibroblast function is altered in a rat model of type II DM.MAIN METHODS:Cardiac fibroblasts were isolated from 14 week old Zucker diabetic and lean control (LC) adult male rat hearts. Fibroblasts were examined for their ability to remodel 3-dimensional collagen matrices, their adhesion, migration and proliferation on collagen and changes in gene expression associated with collagen remodeling.KEY FINDINGS:Cardiac fibroblasts from diabetic animals demonstrated significantly greater ability to contract 3-dimensional collagen matrices compared to cardiac fibroblasts from LC animals. The enhanced contractile behavior was associated with an increase in diabetic fibroblast proliferation and elevated expression of α-smooth muscle actin and type I collagen, suggesting the transformation of diabetic fibroblasts into a myofibroblast phenotype.SIGNIFICANCE:Cardiac fibrosis is a common complication in diabetic cardiomyopathy which may contribute to the observed cardiac dysfunction associated with this disease. Identifying and understanding the changes in fibroblast behavior which contribute to the increased deposition of collagen and other matrix proteins may provide novel therapeutic targets for reducing the devastating effects of diabetes on the heart.

We sought to compare the effects of specific COX 2 inhibitors (Nimesulide) or Prostaglandin D synthase inhibitor (HQL 79) on cardiac remodeling secondary to pressure overload in rats. In vivo left ventricular (LV) structure and function was assessed by pressure/volume catheter at 28 days post surgery in six groups (n ≥ 6 per group): sham‐operated (Sham); untreated pressure overload (PO); Prevention or intervention groups treated with either NIMEsulide (25 mg/kg/d) or HQL 79 (10 mg/kg/d). The prevention strategy was initiated prior to induction of pressure overload and continued to day 14. Intervention treatment strategy started on day 14 post induction of pressure overload and continued till day 28. Both prevention and intervention NIME treatments attenuated the PO induced change in LV mass. Prevention treatment with HQL 79 attenuated this change. End diastolic volume was significantly decreased (by 29%) in PO group compared to Sham. Both treatment strategies with HQL 79 significantly attenuate this change. Prevention treatment with Nimesulide significantly attenuated the change in PO induced increase in total collagen levels, whereas both prevention and intervention treatment with HQL 79 significantly attenuated fibrosis. These findings indicate that cardiac remodeling during pressure overload occurs by Prostaglandin D2 mediated inflammatory pathway and can be attenuated using PGD synthase inhibitors.

This article outlines evidence that advanced glycation end product (AGE) inhibitors and breakers act primarily as chelators, inhibiting metal-catalyzed oxidation reactions that catalyze AGE formation. We then present evidence that chelation is the most likely mechanism by which ACE inhibitors, angiotensin receptor blockers, and aldose reductase inhibitors inhibit AGE formation in diabetes. Finally, we note several recent studies demonstrating therapeutic benefits of chelators for diabetic cardiovascular and renal disease. We conclude that chronic, low-dose chelation therapy deserves serious consideration as a clinical tool for prevention and treatment of diabetes complications.

We hypothesized that pharmacologic blockade of Cyclooxygenase 2(COX 2) using Nimesulide (Nime) prevents maladaptive changes in the LV structure/function secondary to pressure overload. In vivo LV structure/function was assessed by pressure/volume catheter at 14 days post surgery in 3 groups(n ≥ 6 per group); Sham-operated (Sham), Untreated pressure overload (PO) and PO + Nime (10 mg/kg/d). Relative to sham, LV mass index was significantly increased in PO group(P≤0.05). End diastolic volume, an indicator of chamber size, was significantly decreased in the PO animals compared to sham (202 ± 17 vs. 143 ± 16 Sham vs. PO, Nime + PO: 226 ± 9). Hydroxyproline analysis demonstrated collagen levels in PO rats were significantly elevated relative to Sham. Nime treatment significantly prevented the increase in LV mass index, reduction in end diastolic volume and increase in collagen levels. In summary, COX 2 inhibition with Nime prevented the maladaptive changes in the LV after pressure overload. This suggests that acute cardiac remodeling during pathological stress occurs by COX 2 mediated inflammatory pathway and can be attenuated by COX 2 inhibitors. Acknowledging the clinical failure of chronic COX 2 inhibitor use, we propose that acute treatment with COX 2 inhibitors during the initial stages of cardiac remodeling can be beneficial in maintaining normal cardiac structure and function. AHA 09BGIA2090065

The endocannabinoid, anandamide, and cannabinoid CB1 receptors have been implicated in cardiac remodeling. Further, anandamide was shown to be a substrate for the cyclooxygenase‐2 (COX‐2) enzyme which has been linked to cardiac remodeling. This study was designed to determine the expression level of CB1 protein in cardiac remodeling secondary to abdominal aortic constriction model of pressure overload (PO). The effects of the COX‐2 selective inhibitor, Nimesulide (Nime), on the adverse remodeling changes and CB1 protein levels were also further investigated. In vivo LV volume and function were assessed by a pressure/volume catheter at 14 and 28 days post surgery in four groups (n= 4–6 per group) of male SD rats: 1)sham‐operated; 2)untreated PO; 3)prevention treatment with Nime (10mg/kg/d); and 4)intervention treatment with Nime (10mg/kg/d). Relative to sham, LV/body weight index was significantly increased and end diastolic volume was significantly decreased at the 14 and 28 day time points in untreated PO animals. Nime treatment significantly attenuated these changes. Myocardial CB1 protein level was significantly decreased at 14 and 28 days in the untreated PO groups as compared to sham values. Treatment with Nime did not prevent the PO induced decrease in CB1 protein level. The beneficial remodeling changes by COX‐2 inhibition do not appear to be mediated via CB1 receptors.

Chronic activation of the sympathetic nervous system is a key component of cardiac hypertrophy and fibrosis. However, previous studies have provided evidence that also implicate inflammatory cells, including mast cells (MCs), in the development of cardiac fibrosis. The current study investigated the potential interaction of cardiac MCs with the sympathetic nervous system. Eight-week–old male spontaneously hypertensive rats were sympathectomized to establish the effect of the sympathetic nervous system on cardiac MC density, myocardial remodeling, and cytokine production in the hypertensive heart. Age-matched Wistar Kyoto rats served as controls. Cardiac fibrosis and hypertension were significantly attenuated and left ventricular mass normalized, whereas cardiac MC density was markedly increased in sympathectomized spontaneously hypertensive rats. Sympathectomy normalized myocardial levels of interferon-γ, interleukin 6, and interleukin 10, but had no effect on interleukin 4. The effects of norepinephrine and substance P on isolated cardiac MC activation were investigated as potential mechanisms of interaction between the two. Only substance P elicited MC degranulation. Substance P was also shown to induce the production of angiotensin II by a mixed population of isolated cardiac inflammatory cells, including MCs, lymphocytes, and macrophages. These results demonstrate the ability of neuropeptides to regulate inflammatory cell function, providing a potential mechanism by which the sympathetic nervous system and afferent nerves may interact with inflammatory cells in the hypertensive heart.

The ovarian hormone and estrogen receptor dependence of female cardioprotection were investigated using the aortocaval fistula rat model of chronic volume overload. Five groups of female rats were studied: sham‐operated (SHAM), intact fistula (FIST), ovariectomized fistula (FIST‐OX), ovariectomized fistula treated with estrogen (FIST‐OX+EST), and intact fistula rats treated with estrogen receptor blocker ICI 182,780 (FIST+ICI). Cardiac function was assessed temporally for 6 wks using echocardiogram. All rats with fistula had significantly increased cardiac output (119±52 ml/min for FIST‐OX vs. 26±5 ml/min for SHAM, p<0.05). This volume overload induced significant left ventricular (LV) hypertrophy in the fistula groups. All fistula groups developed increased LV internal diastolic dimension (LVIDd), indicating LV dilatation, with the FIST‐OX and FIST+ICI groups having the greatest increases (300% and 290%, respectively, relative to pre‐fistula baselines, p<0.05). This dilatation was associated with significantly reduced ejection fraction. Estrogen treatment attenuated the remodeling developed by ovariectomized females. These data indicate that ovarian hormones are essential for cardioprotection against adverse remodeling in female rats subjected to volume overload, and that this protection is estrogen receptor dependent.Research supported by the American Heart Association (JG ‐ #0435298N).

The potential reciprocal association of pathologic changes in renal and cardiac function as comorbidities has not been well studied. This study examined renal morphology/function as well as left ventricular (LV) systolic/diastolic parameters at two separate time points (e.g. 16 and 36 weeks of age). We hypothesize that the obese ZDF animal of type II diabetes will display systolic/diastolic dysfunction at the earlier time point which will not be associated with renal impairment, however, there will be significant loss of ventricular function at 36 weeks of age which corresponds to the severity of renal pathology. Lean Control (LC) and Zucker Diabetic Fatty (ZDF) rats were assessed for: 1) proteinuria, 2) hydronephrosis, and 3) LV structural/functional parameters at 16 and 36 weeks of age. Evidence of increased urinary albumin and decreased urinary creatinine was paralleled by a marked progression in the severity of hydronephrosis in the 36 week of age ZDF group compared to LC. Relative to LC, end diastolic volume for ZDF animals was significantly increased at 16 and 36 weeks of age without hypertrophy suggestive of LV dilatation. Our results indicate that at 16 weeks of age significant changes in ventricular structure/function already exist without renal insufficiency. At 36 weeks of age there is a strong association between decreased renal function and significant ventricular dysfunction.

dssue from sham-operated (Sham), untreated-fistula (Fist), and bosentan (100 mg'kg-1'day-r)-treated animals Gist + Bos) was analyzed for mast cell density, MMP activity, and myocardial collagen volume ' : ' H;h;; ai''i and 5 days after the qeation of an aortocaval fistula. r When compared with untreated flstulas, bosentan treafrnent prevented the marked increase in LV mast cell density at I day postfistula (3.1 -r , 0.3 vs. 1.3 I 0.3 LV mast cells/mm2, Fist vs. Fist * Bos, P < 0.01). .aOOitionally, the substantial increase in MMP-2 activation in the untreated fistula at I day was prevented following bosentan treatuent : (1.6 + 0.3 vs. 0.9 -f 0.1 arbitrary activity units, Fist vs. Fist * Bos, --._P =__0.01). The marked decrease in collagen volume fraction seen in the Fist group (1.4 + 0.1 vs. 0.8 + O.IVo myocardial tissue, Sham vs. Fist, P < 0.01) was significantly attenuated following bosentan treatment at both the land 5-day time points. Lastly, a 2-wk preventative treatrnent'nith bosentan resulted in significant attenuation of the increase in LV end-systolic and -diastolic volumes conpared with those in untreated fistula hearts. In summary, nonselective ET-l antagonism prevents the acute increases in cardiac mast cell density and MMP activation induced secondary to chronic volume overload. By preventing these events, ET-l antagonism was efficacious in aftenuating ventricular dilatation and limiting the development of structural and functional deficits in the first 2 wk of chronic volume overload. Accordingly, these results are the first to demonstrate that cardiac mast cells are responsive to the endogenous endothelin systen in vivo. Another nouil findine from this studv is that Clronic nonspecific endothelin antagonism iay inadvertently potentiate ETI -mediated signaling.

SUMMARY Previous studies have demonstrated progressive ventricular hypertrophy, dilatation and contractile depression in response to chronic volume overload. Whether this decompensation was related to intrinsic myocyte dysfunction was not clear. The present study evaluated ventricular myocyte function at critical times during the progression of ventricular remodelling induced by volume overload. Chronic volume overload was induced with an infrarenal aortocaval fistula in rats. Myocyte contraction and intracellular Ca 2+ concentrations ([Ca 2+ ] i ) were evaluated using a fura‐2 fluorescence and edge detection system. Protein levels of sarcoplasmic reticulum (SR) Ca 2+ transporters were determined by western blots. Progressive ventricular dilatation developed following creation of the fistula. Although myocyte function in 5 week fistula rats was comparable to that of the control group, myocytes from rats 10 weeks post‐fistula demonstrated significant depression of cell shortening and peak [Ca 2+ ] i . Application of isoproterenol (0.1 µmol/L) was not able to compensate for the functional deficiency in myocytes from 10 week fistula rats. Caffeine (10 mmol/L) induced SR Ca 2+ release, as well as protein expression of SR Ca 2+ ‐ATPase, and ryanodine receptors were reduced in myocytes obtained from the same group of 10 week fistula rats. These data indicate that the transition to heart failure secondary to chronic volume overload is related to depressed myocyte contractility secondary to altered intracellular Ca 2+ homeostasis.

The objective of this study was to investigate the effect a nonselective endothelin-1 (ET-1) receptor antagonist (bosentan) had on the acute myocardial remodeling process including left ventricular (LV) mast cells and matrix metalloproteinase (MMP) activity secondary to volume overload. Additionally, we investigated the overall functional outcome of preventative endothelin receptor antagonism during 14 days of chronic volume overload. LV tissue from sham-operated (Sham), untreated-fistula (Fist), and bosentan (100 mg.kg(-1).day(-1))-treated animals (Fist + Bos) was analyzed for mast cell density, MMP activity, and myocardial collagen volume fraction at 1 and 5 days after the creation of an aortocaval fistula. When compared with untreated fistulas, bosentan treatment prevented the marked increase in LV mast cell density at 1 day postfistula (3.1 +/- 0.3 vs. 1.3 +/- 0.3 LV mast cells/mm2, Fist vs. Fist + Bos, P <or= 0.01). Additionally, the substantial increase in MMP-2 activation in the untreated fistula at 1 day was prevented following bosentan treatment (1.6 +/- 0.3 vs. 0.9 +/- 0.1 arbitrary activity units, Fist vs. Fist + Bos, P <or= 0.01). The marked decrease in collagen volume fraction seen in the Fist group (1.4 +/- 0.1 vs. 0.8 +/- 0.1% myocardial tissue, Sham vs. Fist, P <or= 0.01) was significantly attenuated following bosentan treatment at both the 1- and 5-day time points. Lastly, a 2-wk preventative treatment with bosentan resulted in significant attenuation of the increase in LV end-systolic and -diastolic volumes compared with those in untreated fistula hearts. In summary, nonselective ET-1 antagonism prevents the acute increases in cardiac mast cell density and MMP activation induced secondary to chronic volume overload. By preventing these events, ET-1 antagonism was efficacious in attenuating ventricular dilatation and limiting the development of structural and functional deficits in the first 2 wk of chronic volume overload. Accordingly, these results are the first to demonstrate that cardiac mast cells are responsive to the endogenous endothelin system in vivo. Another novel finding from this study is that chronic nonspecific endothelin antagonism may inadvertently potentiate ET-1-mediated signaling.

. Objective and Design: The purpose of this study was to determine the feasibility of adapting peritoneal and pleural mast cell isolation techniques to recover cardiac mast cells that retain their functional response to the secretagogue, compound 48/80. Methods: Using a novel protocol in rats, viable epicardial mast cells were recovered by aspiration of HBSS injected into the pericardial space. Functionality of these cells was determined by ELISA quantification of histamine release in response to compound 48/80, calcium ionophore A23187 and substance P. Mast cell phenotype was determined based on the presence of chymase and tryptase demonstrated by immunofluorescence, alcian blue-safranin staining, and Western blotting. Results: Mast cells isolated in this manner have low basal rates of histamine release and are highly responsive to these secretagogues. These epicardial mast cells were of the connective tissue type, which is consistent with previous reports characterizing cardiac mast cells isolated from the heart by enzymatic dispersion techniques. Conclusions: This novel pericardial aspiration technique facilitates the straightforward characterization of isolated epicardial mast cell functionality in a controlled in vitro environment, furthering our understanding of their contribution to myocardial disease.

Rats subjected to inhaled diesel exhaust particles (DEP) developed a significant reduction in cardiac function. Histological examination of their hearts demonstrated a significant increase in cardiac mast cell (CMC) density coupled with increased matrix metalloproteinase activity. Pretreatment with a mast cell stabilizing compound prevented these effects on cardiac function indicating that they were mast cell-mediated. However, it was not clear if this mast cell activation was the result of a direct interaction of DEP with CMCs. The purpose of this study was to determine if DEP can directly activate CMCs. CMCs were isolated from rats and exposed to DEP or the mast cell secretagogue 48/80. 48/80 treatment triggered a significant histamine release above that of controls (80.5% and 26.1% of total histamine, respectively). However, exposure to DEP did not result in histamine release above that seen in controls (26.8%). Retained histamine levels were comparable for the control and DEP-exposed cells, ~50 ng/ml. These findings indicate that the DEP effects on cardiac function are not due to a direct action of DEP on CMCs. However, given the ability of endothelin-1 (ET-1) to stimulate mast cell activation in the whole heart and peritoneal mast cells, a DEP-induced ET-1 release from the lungs and subsequent paracrine activation of CMCs represents a plausible mechanism. Support: AHA 0435298N (JDG), EPA RD-83195301-0 (GLB)

BACKGROUND:Recent large randomized, controlled trials (BEST [Beta-blocker Evaluation of Survival Trial], CIBIS-II [Cardiac Insufficiency Bisoprolol Trial II], COPERNICUS [Carvedilol Prospective Randomized Cumulative Survival Study], and MERIT-HF [Metoprolol Randomized Intervention Trial in Congestive Heart Failure]) have addressed the usefulness of beta-blockade in the treatment of advanced heart failure. CIBIS-II, COPERNICUS, and MERIT-HF have shown that beta-blocker treatment with bisoprolol, carvedilol, and metoprolol XL, respectively, reduce mortality in advanced heart failure patients, whereas BEST found a statistically nonsignificant trend toward reduced mortality with bucindolol. We conducted a post hoc analysis to determine whether the response to beta-blockade in BEST could be related to differences in the clinical and demographic characteristics of the study populations. We generated a sample from BEST to resemble the patient cohorts studied in CIBIS-II and MERIT-HF to find out whether the response to beta-blocker therapy was similar to that reported in the other trials. These findings are further compared with COPERNICUS, which entered patients with more severe heart failure. METHODS:To achieve conformity with the entry criteria for CIBIS-II and MERIT-HF, the BEST study population was adjusted to exclude patients with systolic blood pressure <100 mm Hg, heart rate <60 bpm, and age >80 years (exclusion criteria employed in those trials). The BEST comparison subgroup (BCG) was further modified to more closely reflect the racial demographics reported for patients enrolled in CIBIS-II and MERIT-HF. The association of beta-blocker therapy with overall survival and survival free of cardiac death, sudden cardiac death, and progressive pump failure in the BCG was assessed. RESULTS:In the BCG subgroup, bucindolol treatment was associated with significantly lower risk of death from all causes (hazard ratio (HR)=0.77 [95% CI=0.65, 0.92]), cardiovascular death (HR=0.71 [0.58, 0.86]), sudden death (HR=0.77 [0.59, 0.999]), and pump failure death (HR=0.64 [0.45, 0.91]). CONCLUSIONS:Although not excluding the possibility of differences resulting from chance alone or to different properties among beta-blockers, this study suggests the possibility that different heart failure population subgroups may have different responses to beta-blocker therapy.

OBJECTIVES:The risks and benefits of beta-blockade with bucindolol were assessed in heart failure (HF) patients with Class IV symptoms within the Beta-blocker Evaluation of Survival Trial (BEST).BACKGROUND:beta-blockade is accepted therapy for mild to moderate HF, but its safety and efficacy in advanced HF have not been established.METHODS:BEST recruited 2708 HF patients; of these, 226 with Class IV symptoms (n=114 randomized to bucindolol, n=112 to placebo) formed the basis of this study. All-cause death, HF hospitalization, and drug discontinuations occurring early during therapy (< or =6 months) and overall during follow-up were assessed. Compared with Class III, Class IV patients were older and had higher plasma norepinephrine levels, prevalence of coronary disease, S3 gallops, and lower ejection fractions, but characteristics of the 2 Class IV treatment groups were similar.RESULTS:During a mean of 1.6 years, 49% Class IV patients died, and 54% were hospitalized for HF. Bucindolol increased the combined endpoint of death or HF hospitalization within the first 6 months (hazard ratio [HR]=1.7, 95% confidence interval [CI]=1.1-2.7) and did not result in benefit overall (HR=1.2, 95% CI=0.9-1.6). HF hospitalization alone within 6 months was increased by bucindolol (HR=1.7), and an early adverse trend for death was seen (HR=1.6) with no benefit overall (HR=1.1). Bucindolol was discontinued more frequently than placebo for worsening HF (11% versus 4%) and hypotension (3% versus 0%).CONCLUSIONS:Class IV HF patients in BEST were at high risk. Bucindolol did not reduce death or HF hospitalization and was associated with early hazard.

Inhalations of 5 per cent CO 2 for 30 minutes by young diabetic patients produced venular dilation, slowing of erythrocyte velocity, occasional perivenular edema and arteriolar constriction, average arterial blood pH decreases of 0.089 pH units, average plasma bicarbonate content increases of 1.63 mEq./L. and average arterial pCO 2 increases of 11 mm. Hg. The most marked pCO 2 and pH changes occurred in the diabetic subjects with the most degeneration of the small blood vessels.